Collagen, the most abundant protein in mammals, is widely distributed within the body and the rigidity of its rope-like triple helix and assembled fibrils enables it to perform an essential structural role, helping to provide mechanical strength to tissues. The most abundant fibrillar collagens, types I, II and III, occur in skin, bone, cartilage, tendons, ligaments, blood vessels and the vitreous humour of the eye. The more complex non-fibrillar collagens, such as types IV and VI, form two- and three-dimensional networks, supporting the interstitial tissues of the body and being the fundamental component of the basement membranes to which epithelial and endothelial cell layers can attach.
In general, fibrillar collagens contain three separate peptide strands wound around one another to form a triple-helix (Rich A and Crick F H C, J. Mol. Biol., 1961, 3, 483-506). Geometric constraints and the stability of the collagen triple-helix require that every third amino acid be glycine (Gly or G), resulting in a repetitive -GXY- sequence, where X and Y each frequently represent proline (Pro or P) and hydroxyproline (Hyp or O). A collagen triple helix is typically over 300 nm in length and in excess of 1000 amino acids. The fibrils resulting from the assembly of such collagen triple helices exceed 1 μm in length.
In healthy, undamaged tissues, collagen supports the blood vessel wall and its surrounding tissues and is concealed by endothelial cell layers and cannot come in contact with platelets circulating within the bloodstream, which regulate the clotting process. However, damage to the vessel wall, occurring as a consequence of either mechanical trauma or rupture of atherosclerotic plaque in diseased blood vessel walls, may remove the endothelial cell layer and allow collagen to interact with the platelets and other blood plasma proteins, thus activating the platelets for aggregation and adhesion. These processes are essential to the clotting response, and are well understood in the field.
Triple Helical Configuration
Collagen has long fascinated scientists because of the extraordinary structural features and biological importance of these proteins. The study of the structure, stability and function of collagen triple helices has been facilitated by the use of synthetic collagen-related peptides (Feng Y, Melacini G, Taulane J P and Goodman M, J. Am. Chem. Soc., 1996, 118, 10351-10358; Fields G B and Prockop D J, Biopolymers 1996, 40, 345-357 and references cited therein; Holmgren S K, Taylor K M, Bretscher L E and Raines R T, Nature 1998, 392, 666-667; Jenkins C L and Raines R T, Nat. Prod. Rep. 2002, 19, 49-59; and Shah N K, Ramshaw J A M, Kirkpatrick A, Shah C and Brodsky, B. Biochemistry 1996, 35, 10262-10268). For example, the use of synthetic triple-helical peptides comprising specific recognition motifs has allowed receptor-binding properties of the collagens to be investigated in detail. Additionally, the triple-helical conformation of the collagens may be a prerequisite for their recognition by platelet and other collagen receptors. Certain triple-helical sequences, moreover, may directly interact with platelet receptors such as GpVI, including the repeating triplet glycine-proline-hydroxyproline (GPO) sequence. For simple collagen-related peptides, the (GPO)10 (SEQ ID NO: 36) sequence forms thermally stable triple-helices, with a melting temperature of 58-70° C. The hydroxyproline amino acids stabilize the triple-helical structure by facilitating the formation of water mediated hydrogen bonds and by providing stereoelectronic effects.
Furthermore, International Publication Number WO07/052,067 describes a series of short triple-helical collagen peptides covering the type III collagen domain and having platelet adhesion activity based on affinity for the A3 domain of platelet's von Willebrand factor. International Publication WO07/017,671 describes trimer peptides containing GPO repeats which, without crosslinking between the peptides, are able to activate platelets. International Publication WO06/098326 describes a synthetic collagen film prepared from a POG polypeptide and a calcium phosphate compound. Japanese Patent Publication 2005206542 describes collagen tissue structures containing polypeptide sequences Pro-X-Gly and Y-Z-Gly (wherein X and Z represent proline (Pro) and hydroxyproline (Hyp) and Y represents an amino acid residue having a carboxyl group). Japanese Patent Publication 2005126360 describes cosmetic and food compositions containing polypeptide sequence Pro-Y-Gly-Z-Ala-Gly (SEQ ID NO: 37) (wherein Y represents Gln, Asn, Leu, Ile, Val or Ala; and, Z represents Ile or Leu) prepared by solid-phase synthesis for inhibiting collagenase. United States Patent Publication 2003/162941 (equivalent to JP 2003321500) describes collagenous polypeptides with a sequence Pro-Y-Gly (wherein Y represents Pro or Hyp), having a triple helical structure. U.S. Pat. No. 5,973,112 (equivalent to WO99/10381) describes tripeptide collagen mimics of the sequence Xaa-Xbb-Gly (wherein Xaa represents an amino acid residue; and, Xbb represents 4(R)-fluoro-L-proline (Flp), 4(S)-fluoro-L-proline, 4,4-difluoroproline, or an acetyl, mesyl or trifluoromethyl modified hydroxyproline. Collagen mimic (Pro-Flp-Gly)10 (SEQ ID NO: 38) showed increased stability relative to the collagen-related triple helixes Pro-Pro-Gly and Pro-Hyp-Gly.
Self Assembly
Several strategies have been employed in order to induce triple-helical structure formation in isolated collagen ligand sequences (as discussed in U.S. Pat. No. 6,096,863, equivalent of International Publication WO98/007752, and references therein). Triple-helix structure formation in isolated collagen sequences may be induced by adding a number of Gly-Pro-Hyp repeats to both ends of a collagenous sequence. However, even with more than 50% of the peptide sequence consisting of Gly-Pro-Hyp repeats, the resulting triple-helices may not have sufficient thermal stability to survive at physiological conditions. Although substantial stabilization of the triple-helical structure may be achieved with the introduction of covalent links between the C-terminal regions of the three peptide chains, the large size (90-125 amino acid residues) of the resulting “branched” triple-helical peptide compounds make them difficult to synthesize and purify (as discussed in U.S. Pat. No. 6,096,863 and references therein). While oligomerized CRPs, via dendrimer assembly or covalent crosslinking, may effectively induce platelet aggregation without being immobilized, less organized CRPs such as those having a (POG)10 (SEQ ID NO: 34) sequence, lack this property (Rao G H R, Fields C G, White J G and Fields G B, J. Biol. Chem. 1994, 269, 13899-13903; Morton L F, Hargreaves P G, Farndale R W, Young R D and Barnes M J, Biochem. J. 1995, 306, 337-344; Knight C G, Morton L F, Onley D J, Peachey A R, Ichinohe T, Okuma M, Farndale R W and Barnes M J. Cardiovasc. Res. 1999, 41, 450-457). The availability and usefulness of CRPs capable of self-assembly has been dependent on the ease of their preparation, the simplicity and stability of the CRP structure and the potential for aggregation activity. Although the synthesis may be challenging and relatively complex, micrometer-scale CRP-based materials were obtained from the self-assembly of covalently attached triple-stranded entities by employing a cysteine knot (Koide T, Homma D L, Asada S and Kitagawa K, Bioorg. Med. Chem. Lett. 2005, 15, 5230-5233; and, Kotch F and Raines R T, Proc. Natl. Acad. Sci. USA 2006, 103, 3028-3033).
Thus, what is still needed are simplified approaches to building a collagen-like structural motif that facilitates peptide alignment and fibril initiation and propagation. Specifically, what is needed are relatively short, single-strand CRPs that are easily synthesized and are capable of non-covalent self-assembly into trimers having collagen-mimetic properties.